Wu Xunwei, Li Shaohua, Chrostek-Grashoff Anna, Czuchra Aleksandra, Meyer Hannelore, Yurchenco Peter D, Brakebusch Cord
University of Copenhagen, Institute of Molecular Pathology, Copenhagen, Denmark.
Dev Dyn. 2007 Oct;236(10):2767-78. doi: 10.1002/dvdy.21309.
To study the role of Cdc42 in the establishment of epithelial polarity during mammalian development, we generated murine Cdc42-null embryonic stem cells and analyzed peri-implantation development using embryoid bodies (EBs). Mutant EBs developed endoderm and underlying basement membrane, but exhibited defects of cell polarity, cell-cell junctions, survival, and cavitation. These defects corresponded to a decreased phosphorylation and membrane localization of aPKC, a reduced phosphorylation of GSK3beta, and a diminished activity of Rac1. However, neither Rac1 nor the kinase function of GSK3beta seem to contribute to cell polarization and cell-cell contacts. In contrast, EBs expressing dominant-negative (dn) PKCzeta mimicked well the phenotype of Cdc42-null EBs, suggesting a major role of aPKC in mediating cell polarization downstream of Cdc42. Finally, aggregation experiments with endodermal cell lines suggested that Cdc42 might affect formation of adherens and tight junctions by PKCzeta-dependent regulation of the protein levels of p120 catenin and E-cadherin.
为了研究Cdc42在哺乳动物发育过程中上皮极性建立中的作用，我们生成了小鼠Cdc42基因敲除胚胎干细胞，并使用胚状体（EBs）分析植入前发育情况。突变型EBs发育出内胚层和其下方的基底膜，但表现出细胞极性、细胞间连接、存活和空泡化方面的缺陷。这些缺陷与非典型蛋白激酶C（aPKC）的磷酸化和膜定位降低、糖原合成酶激酶3β（GSK3β）的磷酸化减少以及Rac1活性降低相对应。然而，Rac1和GSK3β的激酶功能似乎均未对细胞极化和细胞间接触产生影响。相反，表达显性负性（dn）PKCζ的EBs很好地模拟了Cdc42基因敲除EBs的表型，表明aPKC在介导Cdc42下游的细胞极化中起主要作用。最后，内胚层细胞系的聚集实验表明，Cdc42可能通过PKCζ依赖的方式调节p120连环蛋白和E-钙黏蛋白的蛋白水平，从而影响黏着连接和紧密连接的形成。
